Physical interactions of the peroxisomal targeting signal 1 receptor pex5p, studied by fluorescence correlation spectroscopy.
AuthorsWang D, Visser NV, Veenhuis M, van der Klei IJ
JournalJ Biol Chem
PubMed ID12930827
'We have studied Hansenula polymorpha Pex5p and Pex8p using fluorescence correlation spectroscopy (FCS). Pex5p is the Peroxisomal Targeting Signal 1 (PTS1) receptor and Pex8p is an intraperoxisomal protein. Both proteins are essential for PTS1 protein import and have been shown to physically interact. We used FCS to analyze the molecular ... More
Toxicity of organic fluorophores used in molecular imaging: literature review.
AuthorsAlford R, Simpson HM, Duberman J, Hill GC, Ogawa M, Regino C, Kobayashi H, Choyke PL,
JournalMol Imaging
PubMed ID20003892
'Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of ... More
The photon counting histogram in fluorescence fluctuation spectroscopy.
AuthorsChen Y, Müller JD, So PT, Gratton E
JournalBiophys J
PubMed ID10388780
'Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence ... More
Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization.
AuthorsHellen EH, Burghardt TP
JournalBiophys J
PubMed ID8011921
'The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed ... More
Fluorogenic substrate [Ala-Pro]2-cresyl violet but not Ala-Pro-rhodamine 110 is cleaved specifically by DPPIV activity: a study in living Jurkat cells and CD26/DPPIV-transfected Jurkat cells.
AuthorsBoonacker E, Elferink S, Bardai A, Fleischer B, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID12810846
'Fluorogenic substrates [Ala-Pro](2)-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that ... More
Monolithic integration of optical waveguides for absorbance detection in microfabricated electrophoresis devices.
AuthorsMogensen KB, Petersen NJ, Hübner J, Kutter JR
JournalElectrophoresis
PubMed ID11700723
'The fabrication and performance of an electrophoretic separation chip with integrated optical waveguides for absorption detection is presented. The device was fabricated on a silicon substrate by standard microfabrication techniques with the use of two photolithographic mask steps. The waveguides on the device were connected to optical fibers, which enabled ... More
Multiplexed detection of protein-peptide interaction and inhibition using capillary electrophoresis.
AuthorsYang P, Whelan RJ, Mao Y, Lee AW, Carter-Su C, Kennedy RT
JournalAnal Chem
PubMed ID17297974
'High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain ... More
Stacking due to ionic transport number mismatch during sample sweeping on microchips.
AuthorsLiu Y, Foote RS, Jacobson SC, Ramsey JM
JournalLab Chip
PubMed ID15791345
Sample stacking can occur in isoconductive buffer systems as a result of ion transport mismatches that cause changes in buffer conductivity during electrophoresis. Fluorescence imaging was used to examine this effect in the sweeping of hydrophobic dyes with sodium dodecyl sulfate (SDS) on microchips. Imaging revealed the occurrence of a ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
A homogeneous, nonradioactive high-throughput fluorogenic protein kinase assay.
AuthorsKupcho K, Somberg R, Bulleit B, Goueli SA
JournalAnal Biochem
PubMed ID12758259
Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, ... More
We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence ... More
Shah convolution Fourier transform detection: multiple-sample injection technique.
AuthorsKwok YC, Manz A
JournalElectrophoresis
PubMed ID11288888
For the direct measurement of electrophoretic mobility, multiple-point (Shah function) detected, time-domain detector signals were converted into frequency-domain plots by means of Fourier transformation. Multiple sample plugs (up to a maximum of three) were introduced into the separation channel and the resultant time-domain signals were then Fourier-transformed. The multiple-sample injection ... More
We introduce dual-color time-integrated fluorescence cumulant analysis (TIFCA) to analyze fluorescence fluctuation spectroscopy data. Dual-color TIFCA utilizes the bivariate cumulants of the integrated fluorescent intensity from two detection channels to extract the brightness in each channel, the occupation number, and the diffusion time of fluorophores simultaneously. Detecting the fluorescence in ... More
A homogeneous, nonradioactive high-throughput fluorogenic protein phosphatase assay.
AuthorsKupcho K, Hsiao K, Bulleit B, Goueli SA
JournalJ Biomol Screen
PubMed ID15140384
Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive ... More
Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates.
AuthorsGanesh S, Klingel S, Kahle H, Valet G
JournalCytometry
PubMed ID7587721
Aminopeptidases (AP) are ubiquitously occurring, nonspecific exopeptidases involved in protein degradation. They cleave the N-terminal amino acid of peptides and occur in practically all mammalian cells and tissues. Physiological and pathological processes such as metastasis of tumors and inflammation have been thought to involve changes in AP activities. Determination of ... More
Intracellular accumulation of rhodamine 110 in single living cells.
AuthorsJeannot V, Salmon JM, Deumié M, Viallet P
JournalJ Histochem Cytochem
PubMed ID9071322
To gain a better understanding of the internalization of rhodamines, vital staining of living cells in situ by two different rhodamines, R110 and R123, was studied by microfluorometry. These dyes differ strongly in their lipophilic properties because of differences in charge distribution. Microspectrofluorometry was used to study the fluorescence emission ... More
Examination of the functional activity of P-glycoprotein in the rat placental barrier using rhodamine 123.
AuthorsPavek P, Staud F, Fendrich Z, Sklenarova H, Libra A, Novotna M, Kopecky M, Nobilis M, Semecky V
JournalJ Pharmacol Exp Ther
PubMed ID12626638
Rhodamine 123 (Rho123), a model substrate of P-glycoprotein (P-gp), was used to evaluate the functional activity of P-gp efflux transporter in the rat placental barrier. The dually perfused rat-term placenta method was used. In our experiments, the materno-fetal transplacental passage of Rho123 did not meet the criteria of the first-order ... More